Stress and burnout syndrome in health-care providers treating dengue infection: A cross-sectional study.

INTRODUCTION: Increased prevalence of dengue fever had led to increase stress in providing optimal care for patients. This has been identified as a potential factor that may lead to negative health effects on medical doctors. This study was designed to review the prevalence and associated factors of burnout syndrome (including depression, anxiety, and stress level) among clinicians in the setting of increasing cases of dengue in Malaysia. METHODS: A cross-sectional, multi-centre study was carried out among doctors in contact with patients with dengue infection from four major hospitals in Malaysia in 2015 using Maslach Burnout Inventory and DASS-21 questionnaire. RESULTS: A total of 313 respondents were included in this study with 15.9% of the respondents experiencing high burnout syndrome. Long working hours, depression, anxiety, and stress were significantly associated with high degree of burnout syndrome (p<0.05). However, number of dengue cases reviewed was not significantly associated with the degree of burnout syndrome. Depression and stress were among factors identified as the predictors for burnout syndrome. CONCLUSION: High degree of burnout syndrome among clinicians with significant correlations with symptoms of depression and stress will require early identification to enable early measures to resolve, as well as prevent it. Future studies with more hospitals involvement should be conducted to establish the relationship between the degree of burnout syndrome and prevalence of dengue infection.

Gene expression profiles in circulating tumor cells to predict prognosis in metastatic breast cancer patients.

BACKGROUND: A circulating tumor cell (CTC) count is an established prognostic factor in metastatic breast cancer (MBC). Besides enumeration, CTC characterization promises to improve outcome prediction and treatment guidance. Having shown the feasibility of quantifying clinically relevant mRNA transcripts in CTCs, we determined the prognostic value of CTC gene expression in MBC. PATIENTS AND METHODS: CTCs were isolated and enumerated from blood of 197 MBC patients who were about to start first-line systemic therapy. Of these, 180 were assessable for quantification of mRNA expression by RT-qPCR in relation to time-to-treatment failure (TTF). A prognostic CTC gene profile was generated by leave-one-out cross validation in a 103 patient discovery set and validated in 77 patients. Additionally, all 180 patients were randomly divided into two equal sets to discover and validate a second prognostic profile. RESULTS: CTC count predicted for TTF at baseline {≥5 versus <5 CTCs/7.5 ml blood, hazard ratio (HR) 2.92 [95% confidence interval (CI) 1.71-4.95] P < 0.0001}. A 16-gene CTC profile was generated in the first discovery set, which identified patients with death or TTF <9 months versus those with a better outcome. In multivariate analysis, the 16-gene profile was the only factor associated with TTF [HR 3.15 (95% CI 1.35-7.33) P 0.008]. Validation of this profile in the independent patient set pointed into the same direction, but was not statistically significant. A newly generated 8-gene profile showed similarly favorable test characteristics as the 16-gene profile, but did not significantly pass validation either. CONCLUSION: A 16-gene CTC profile was identified, which provided prognostic value on top of CTC count in MBC patients. However, validation of this profile in an independent cohort, nor of a second profile, reached statistical significance, underscoring the need to further fine-tune the still promising approach of CTC characterization.

High protein expression of EZH2 is related to unfavorable outcome to tamoxifen in metastatic breast cancer.

BACKGROUND: Metastatic breast cancer (MBC) is a highly heterogeneous disease with great differences in outcome to both chemo- and endocrine therapy. Better insight into the mechanisms underlying resistance is essential to better predict outcome to therapy and to obtain a more tailored treatment approach. We have previously described that increased mRNA expression levels of Enhancer of Zeste homolog (EZH2) are associated with worse outcome to tamoxifen therapy in MBC. Here, we explored whether this is also the case for EZH2 protein expression. PATIENTS AND METHODS: A tissue microarray (TMA) was created using formalin-fixed, paraffin-embedded estrogen receptor (ER)-positive primary breast tumor tissues of 250 MBC patients treated with first-line tamoxifen. Quantity and intensity of EZH2 expression were determined by immunohistochemistry (IHC) and both were used to generate and group scores according to a previously described method for scoring EZH2. RESULTS: In total, 116 tumors (46%) were considered to be EZH2 positive. The presence of EZH2 protein expression was significantly associated with progression-free survival (PFS) in both univariate [hazard ratio (HR) 1.51, 95% confidence interval (CI) 1.17-1.97, P = 0.002] and multivariate analysis including traditional factors associated with tamoxifen outcome (HR 1.41, 95% CI 1.06-1.88, P = 0.017). Considering quantity irrespective of intensity, tumors with >50% EZH2-positive cells had the worst PFS (HR 2.15, 95% CI 1.42-3.27, P < 0.001), whereas intensity alone did not show a significant association with PFS. Application of other methods of scoring EZH2 positivity resulted in a similar significant association between the amount of EZH2 positive cells and PFS. CONCLUSION: In addition to EZH2 mRNA levels, these results suggest that protein expression of EZH2 can be used as a marker to predict outcome to tamoxifen therapy. This provides new rationale to explore EZH2 inhibition in the clinical setting and increases the possibilities for a more personalized treatment approach in MBC patients.

Metabolic equivalent determination in the cultural dance of hula.

Ethnic minorities share an unequal burden of cardiometabolic syndrome. Physical activity (PA) has been shown to be an important factor for improving the outcomes of these diseases. While metabolic equivalents (METs) have been calculated for diverse activities, most cultural activities have not been evaluated. Hula, the traditional dance of Native Hawaiians, is practiced by men and women of all ages but its MET value is unknown. To our knowledge, this is the first scientific evaluation of energy expenditure of hula. 19 competitive hula dancers performed 2 dance sets of low- and high-intensity hula. METs were measured with a portable indirect calorimetry device. Mean and standard deviations were calculated for all the variables. A 2-way ANOVA was conducted to identify differences for gender and intensity. The mean MET were 5.7 (range 3.17-9.77) and 7.55 (range 4.43-12.0) for low-intensity and high-intensity, respectively. There was a significant difference between intensities and no significant difference between genders. This study demonstrates that the energy expenditure of both low- and high-intensity hula met the recommended guidelines for moderate and vigorous intensity exercise, respectively, and that hula can be utilized as a prescribed PA.

Association of bone turnover markers with mortality in men referred to coronary angiography.

UNLABELLED: We aimed to examine the association of fatal events with osteocalcin (OC) and beta-crosslaps (ß-CTX) levels in men. We observed a U-shaped association of OC and ß-CTX levels with fatal events in a large cohort of men at high cardiovascular risk. INTRODUCTION: Accumulating evidence suggests an association of low OC levels with metabolic disturbances. Whether OC levels are related to fatal events is, however, less clear. Further, high ß-CTX levels are linked to increased mortality. We aimed to examine the association of fatal events with both OC and ß-CTX in men. METHODS: We measured OC and ß-CTX in 2,271 men referred to coronary angiography (1997-2000). RESULTS: We observed a U-shaped association of OC and ß-CTX with fatal events. Crude hazard ratios (HRs) for all-cause and non-cardiovascular mortality in the highest OC quintile were 1.38 (1.04-1.83) and 1.47 (0.89-2.40), respectively, and 2.11 (1.61-2.75) and 2.06 (1.29-3.29) for men in the lowest compared to the third OC quintile. In multivariate-adjusted models, HRs for all-cause, and non-cardiovascular mortality in the lowest OC quintile were 1.63 (1.23-2.16) and 1.79 (1.10-2.92), respectively, compared to the third OC quintile, whereas the association of high OC with mortality lost its significance. Crude and multivariate-adjusted HRs for cardiovascular mortality in the lowest OC quintile compared to the third OC quintile were 2.08 (1.49-2.90) and 1.74 (1.24-2.46), respectively. Moreover, high as well as low ß-CTX levels were independently associated with all-cause (quintile 1 vs. quintile 3: HR 1.42 (1.05-1.92); quintile 5 vs. quintile 3: HR 1.79 (1.31-2.45)) and cardiovascular mortality (quintile 1 vs. quintile 3: HR 1.55 (1.05-2.28); quintile 5 vs. quintile 3: HR 1.85 (1.23-2.77)). CONCLUSIONS: We observed a U-shaped association of OC and ß-CTX with fatal events in a large cohort of men at high cardiovascular risk.

BCAR4 induces antioestrogen resistance but sensitises breast cancer to lapatinib.

BACKGROUND: High BCAR4 and ERBB2 mRNA levels in primary breast cancer associate with tamoxifen resistance and poor patient outcome. We determined whether BCAR4 expression sensitises breast cancer cells to lapatinib, and identifies a subgroup of patients who possibly may benefit from ERBB2-targeted therapies despite having tumours with low ERBB2 expression. METHODS: Proliferation assays were applied to determine the effect of BCAR4 expression on lapatinib treatment. Changes in cell signalling were quantified with reverse-phase protein microarrays. Quantitative reverse-transcriptase polymerase chain reaction (RT-PCR) of ERBB2 and BCAR4 was performed in 1418 primary breast cancers. Combined BCAR4 and ERBB2 mRNA levels were evaluated for association with progression-free survival (PFS) in 293 oestrogen receptor-α (ER)-positive patients receiving tamoxifen as first-line monotherapy for recurrent disease. RESULTS: BCAR4 expression strongly sensitised ZR-75-1 and MCF7 breast cancer cells to the combination of lapatinib and antioestrogens. Lapatinib interfered with phosphorylation of ERBB2 and its downstream mediators AKT, FAK, SHC, STAT5, and STAT6. Reverse transcriptase-PCR analysis showed that 27.6% of the breast cancers were positive for BCAR4 and 22% expressed also low levels of ERBB2. The clinical significance of combining BCAR4 and ERBB2 mRNA status was underscored by the finding that the group of patients having BCAR4-positive/ERBB2-low-expressing cancers had a shorter PFS on tamoxifen treatment than the BCAR4-negative group. CONCLUSION: This study shows that BCAR4 expression identifies a subgroup of ER-positive breast cancer patients without overexpression of ERBB2 who have a poor outcome and might benefit from combined ERBB2-targeted and antioestrogen therapy.

High miR-26a and low CDC2 levels associate with decreased EZH2 expression and with favorable outcome on tamoxifen in metastatic breast cancer.

For patients with metastatic breast cancer, we previously described that increased EZH2 expression levels were associated with an adverse outcome to tamoxifen therapy. Main objective of the present study is to investigate miR-26a and miR-101 levels, which both target EZH2, for their association with molecular pathways and with efficacy of tamoxifen as first-line monotherapy for metastatic breast cancer. Expression levels were measured using quantitative Real-Time Polymerase Chain Reaction (qRT-PCR) in primary breast cancer specimens of 235 estrogen receptor-α (ER)-positive patients. Pathway analysis was performed on microarray data available for 65 of these tumors. Logistic regression and Cox uni- and multivariate analysis were performed to relate expression levels with clinical benefit and time to progression (TTP). Increasing levels of miR-26a were significantly (P < 0.005) associated with both clinical benefit and prolonged TTP, whereas miR-101 was not. Cell cycle regulation and CCNE1 and CDC2 were the only significant overlapping pathway and genes differentially expressed between tumors with high and low levels of miR-26a and EZH2, respectively. In addition, increasing mRNA levels of CCNE1 (P < 0.05) and CDC2 (P < 0.001) were related to poor outcome. Multivariate analysis revealed miR-26a and CDC2 as an optimal set of markers associated with outcome on tamoxifen therapy, independently of traditional predictive factors. To summarize, only miR-26a levels are related with treatment outcome. Cell cycle regulation is the only overlapping pathway linked to miR-26a and EZH2 levels. Low mRNA levels of EZH2, CCNE1, and CDC2, and high levels of miR-26a are associated with favorable outcome on tamoxifen.

Nanoparticle delivery of mycophenolic acid upregulates PD-L1 on dendritic cells to prolong murine allograft survival.

Conventional immunosuppressive drug delivery requires high systemic drug levels to provide therapeutic benefit, but frequently results in toxic side effects. Novel drug delivery methods, such as FDA-approved poly(lactic-co-glycolic acid) (PLGA) nanoparticles (NPs), are promising drug delivery platforms to reduce drug doses and minimize toxicity. Using murine models of skin transplantation, we investigated whether PLGA NPs would effectively deliver mycophenolic acid (MPA), a common clinical immunosuppressant, and avoid the toxicity of conventional drug delivery. We found that intermittent treatment with NPs encapsulated with MPA (NP-MPA) resulted in a significant extension of allograft survival than intermittent conventional MPA treatment even though the concentration of MPA within NP-MPA was a 1000-fold lower than conventional drug. Importantly, recipients who were administered NP-MPA intermittently avoided drug toxicity, whereas those treated with daily conventional drug manifested cytopenias. Dendritic cells (DCs) endocytosed NP-MPA to upregulate programmed death ligand-1 (PD-L1) and displayed a decreased ability to prime alloreactive T cells. Importantly, the ability of NP-MPA to promote allograft survival was partly PD-L1 dependent. Collectively, this study indicates that NPs are potent drug delivery tools that extend allograft survival without drug toxicity.

Decreased expression of EZH2 is associated with upregulation of ER and favorable outcome to tamoxifen in advanced breast cancer.

The purpose of this study is to investigate EZH2 in a large series of breast cancer patients for its prognostic and predictive value, and to evaluate its functional role in treatment response in vitro. EZH2 levels were measured using quantitative Real-Time Polymerase Chain Reaction (qRT-PCR) in primary breast cancer specimens and related to clinicopathologic factors and disease outcome. EZH2 expression was downregulated with siRNAs in MCF7, to assess expression alterations of putative EZH2 downstream genes and to determine cell numbers after treatment with the anti-estrogen ICI 164384. In 688 lymph node-negative patients who did not receive adjuvant systemic therapy, EZH2 was not significantly correlated with metastasis-free survival (MFS). In 278 patients with advanced disease treated with first-line tamoxifen monotherapy, the tertile with highest EZH2 levels was associated with the lowest clinical benefit (OR = 0.48; P = 0.02) and with a shorter progression-free survival (PFS) in both univariate (HR = 1.80; P < 0.001) and multivariate analysis, including traditional factors (HR = 1.61; P = 0.004). In vitro, EZH2 silencing in MCF7 caused a 38% decrease in cell numbers (P < 0.001) whereas ICI 164384 treatment resulted in a 25% decrease (P < 0.001) compared to controls. Combining EZH2 silencing with ICI treatment reduced cell numbers with 67% (P < 0.001) compared to control conditions. EZH2 downregulation was associated with an almost two-fold upregulation of the estrogen receptor alpha (ER) (P = 0.001). In conclusion, EZH2 has no prognostic value in breast cancer. High levels of EZH2 are associated with poor outcome to tamoxifen therapy in advanced breast cancer. Downregulated EZH2 leads to upregulation of the ER and better response to anti-estrogens.

Bridging the communication gap between radiographers and patients to improve chest radiography image acquisition: A multilingual solution in the COVID-19 pandemic.

INTRODUCTION: A chest X-ray (CXR), taken in full inspiration, is important to ensure pathology in the lungs will not be missed. To achieve this, effective communication on breathing instructions for patients is crucial. During the COVID-19 pandemic, radiographers in Sengkang General Hospital (SKH) were challenged when performing CXR for the patients whose native language is not English. Most of these patients were foreign workers living in the same dormitory which had formed the largest COVID-19 cluster in Singapore. These dormitory residents found it difficult to understand and adhere to breathing instructions, resulting in a suboptimal degree of inspiration when the CXRs were taken. This may ultimately affect the diagnostic value of the radiographs. This paper aims to share and evaluate how radiographers tackled this issue and continued to acquire fully-inspired CXR for the dormitory residents despite the language barrier. METHODS: Using a combination of online survey and retrospective analysis of the rejection rates of CXR done over the period of early April to early June, a team of radiographers evaluated the effectiveness of using audio recordings in managing the issue of not achieving a fully inspired CXR for patients due to language barrier. RESULTS: The rejection rate for CXR due to suboptimal inspiration decreased from 26% to 9% upon implementation of the audio recordings. 92.3% of the CXRs taken within this period also fulfilled the criteria of a fully-inspired CXR, as evidenced by having at least 9 posterior ribs seen above the right hemi-diaphragm. Survey results found a fairly balanced number of radiographers who agreed and disagreed that a fully-inspired CXR was achieved for most of their patients after utilisation of translation manuals and audio recordings. CONCLUSION: After the implementation of audio recordings, the decrease in rejection rate of CXR and an audit which demonstrated that CXR quality was upheld had proven that the radiographers successfully achieved fully-inspired CXR for suspected COVID-19 patients. This confirmed that using pre-recorded audio instructions was an efficient intervention albeit being a one-way communication, leads to more accurate imaging results, aligning with existing literature on communication experiences between radiographers and patients. Moreover, the decreased rejection rate of CXRs had increased department efficiency consequently reducing departmental expenses in the long run. IMPLICATIONS OF PRACTICE: Given that we have an ageing population and the vast majority of the elderly converse in their various dialects, positive feedback from radiographers presented opportunities to expand the translation manual and audio recordings to include local dialects. These can be seamlessly integrated in CXR and other procedures in the hospital setting. To ensure that the translations are culturally sensitive, attention should be paid to the translation process of instructions into other languages and local dialects by enlisting the help of native speakers.

Relevance of BCAR4 in tamoxifen resistance and tumour aggressiveness of human breast cancer.

BACKGROUND: Breast cancer anti-oestrogen resistance 4 (BCAR4) was identified in a search for genes involved in anti-oestrogen resistance in breast cancer. We explored whether BCAR4 is predictive for tamoxifen resistance and prognostic for tumour aggressiveness, and studied its function. METHODS: BCAR4 mRNA levels were measured in primary breast tumours, and evaluated for association with progression-free survival (PFS) and clinical benefit in patients with oestrogen receptor (ERα)-positive tumours receiving tamoxifen as first-line monotherapy for advanced disease. In a separate cohort of patients with lymph node-negative, ERα-positive cancer, and not receiving systemic adjuvant therapy, BCAR4 levels were evaluated for association with distant metastasis-free survival (MFS). The function of BCAR4 was studied with immunoblotting and RNA interference in a cell model. RESULTS: Multivariate analyses established high BCAR4 mRNA levels as an independent predictive factor for poor PFS after start of tamoxifen therapy for recurrent disease. High BCAR4 mRNA levels were associated with poor MFS and overall survival, reflecting tumour aggressiveness. In BCAR4-expressing cells, phosphorylation of v-erb-b2 erythroblastic leukaemia viral oncogene homolog (ERBB)2, ERBB3, and their downstream mediators extracellular signal-regulated kinase 1/2 and v-akt murine thymoma viral oncogene homolog (AKT) 1/2, was increased. Selective knockdown of ERBB2 or ERBB3 inhibited proliferation, confirming their role in BCAR4-induced tamoxifen resistance. CONCLUSION: BCAR4 may have clinical relevance for tumour aggressiveness and tamoxifen resistance. Our cell model suggests that BCAR4-positive breast tumours are driven by ERBB2/ERBB3 signalling. Patients with such tumours may benefit from ERBB-targeted therapy.

Stem cell-like transcriptional reprogramming mediates metastatic resistance to mTOR inhibition.

Inhibitors of the mechanistic target of rapamycin (mTOR) are currently used to treat advanced metastatic breast cancer. However, whether an aggressive phenotype is sustained through adaptation or resistance to mTOR inhibition remains unknown. Here, complementary studies in human tumors, cancer models and cell lines reveal transcriptional reprogramming that supports metastasis in response to mTOR inhibition. This cancer feature is driven by EVI1 and SOX9. EVI1 functionally cooperates with and positively regulates SOX9, and promotes the transcriptional upregulation of key mTOR pathway components (REHB and RAPTOR) and of lung metastasis mediators (FSCN1 and SPARC). The expression of EVI1 and SOX9 is associated with stem cell-like and metastasis signatures, and their depletion impairs the metastatic potential of breast cancer cells. These results establish the mechanistic link between resistance to mTOR inhibition and cancer metastatic potential, thus enhancing our understanding of mTOR targeting failure.

Bcar1/p130Cas protein and primary breast cancer: prognosis and response to tamoxifen treatment.

BACKGROUND: The product of the Bcar1/p130Cas (breast cancer resistance/p130Crk-associated substrate) gene causes resistance to antiestrogen drugs in human breast cancer cells in vitro. To investigate its role in clinical breast cancer, we determined the levels of Bcar1/p130Cas protein in a large series of primary breast carcinomas. METHODS: We measured Bcar1/p130Cas protein in cytosol extracts from 937 primary breast carcinomas by western blot analysis. The levels of Bcar1/p130Cas protein were tested for associations and trends against clinicopathologic and patient characteristics, the lengths of relapse-free survival and overall survival (n = 775), and the efficacy of first-line treatment with tamoxifen for recurrent or metastatic disease (n = 268). RESULTS: Bcar1/p130Cas levels in primary tumors were associated with age/menopausal status and the levels of estrogen receptor and progesterone receptor. In univariate survival analysis, higher Bcar1/p130Cas levels were associated with poor relapse-free survival and overall survival (both two-sided P =.04; log-rank test for trend). In multivariate analysis, a high level of Bcar1/p130Cas was independently associated with poor relapse-free survival and overall survival. The response to tamoxifen therapy in patients with recurrent disease was reduced in patients with primary tumors that expressed high levels of Bcar1/p130Cas. In multivariate analysis for response, Bcar1/p130Cas was independent of classical predictive factors, such as estrogen receptor status, age/menopausal status, disease-free interval, and dominant site of relapse. CONCLUSION: Patients with primary breast tumors expressing a high level of Bcar1/p130Cas protein appear to experience more rapid disease recurrence and have a greater risk of (intrinsic) resistance to tamoxifen therapy. Thus, measurement of Bcar1/p130Cas may provide useful prognostic information for patients with primary or metastatic breast cancer.

Urokinase-type plasminogen activator and its inhibitor PAI-1: predictors of poor response to tamoxifen therapy in recurrent breast cancer.

BACKGROUND: Urokinase-type plasminogen activator (uPA) is a proteolytic enzyme thought to be involved in processes leading to tumor cell invasion of surrounding tissues. Its activity during metastasis may be regulated by an inhibitor, PAI-1. Previous work has shown that high levels of uPA and PAI-1 are associated with poor prognosis in primary breast cancers. PURPOSE: In this pilot study, we explored possible associations between the expression levels of uPA or PAI-1 and the efficacy of tamoxifen treatment in breast cancer patients with relapsed disease. METHODS: Levels of uPA, PAI-1, estrogen receptor (ER), and progesterone receptor (PgR) were assayed in cytosolic extracts derived from the primary breast tumors of 235 tamoxifennaive patients who had recurrent disease. The extracts were classified as positive or negative for each assayed factor. In some analyses, ER and PgR levels were evaluated together. In these analyses, three ER/PgR subsets were defined: low, intermediate, and high. All patients in the study received tamoxifen therapy upon relapse (median follow-up, 57 months). Association of the tested factors with the response to tamoxifen treatment was studied by logistic regression analysis. Association of the factors with progression-free and overall survival was further evaluated by Cox univariate and multivariate regression analyses. RESULTS: Patients with uPA-negative tumors exhibited a better response (tumor regression or stable disease, maintained for more than 6 months) to tamoxifen treatment than those with uPA-positive tumors (51% versus 26% response; odds ratio [OR] = 0.34; 95% confidence interval [CI] = 0.18-0.65). The response rate was also better for patients with PAI-1-negative tumors than for those with PAI-1-positive tumors (49% versus 35% response; OR = 0.57; 95% CI = 0.32-1.01). In addition, patients with uPA-positive or PAI-1-positive tumors showed shorter progression-free survival (P = .001 and P < .05, respectively) and total survival after relapse (P = .005 and P < .005, respectively). When patients were stratified by ER/PgR status, the only statistically significant association between uPA levels and reduced tamoxifen response was seen in the subset whose tumors possessed intermediate levels of ER/PgR (16% response in uPA-positive versus 60% response in uPA-negative tumors; OR = 0.13; 95% CI = 0.04-0.41). Overall, uPA status appeared independent of association with ER/PgR status in its ability to predict response to tamoxifen treatment. The association of PAI-1 expression and the response to tamoxifen was less pronounced when patients were stratified by ER/PgR status. CONCLUSION: Measurement of primary breast tumor uPA levels may be useful in predicting the overall response of metastatic disease to tamoxifen therapy.

Thymidine kinase and thymidylate synthase in advanced breast cancer: response to tamoxifen and chemotherapy.

Thymidylate synthase (TS) is a crucial target for 5-fluorouracil (5-FU) in the de novo pathway of pyrimidine synthesis, which is necessary for DNA synthesis. Thymidine kinase (TK) plays a key role in the complementary or alternative salvage pathway of pyrimidine synthesis in acute or pathological tissue stress. In the present study, the activity levels of TS and TK were determined in 257 primary breast tumors of patients who received tamoxifen as first-line systemic therapy after diagnosis of advanced disease. In 155 (60%) responding patients, the median response duration was 23 months for tumors with low TK activity, 15 months for tumors with intermediate TK activity, and 13 months for tumors with high TK activity (P = 0.003). In Cox multivariate analysis corrected for classical predictive factors including estrogen receptor and progesterone receptor, patients with intermediate and high levels of TK activity in their tumors showed a rapid disease progression (P = 0.0002) and an early death (P = 0.002) after start of tamoxifen treatment. Tumor TS activity levels were not significantly associated with the efficacy of tamoxifen treatment. In 121 patients who became resistant to tamoxifen or additional endocrine treatments and who received 5-FU-containing polychemotherapy, tumor TK activity was not significantly related to the efficacy of chemotherapy. Of the 13 patients with low tumor TS activity, only 1 (8%) responded favorably, whereas 46% (43 of 93) of those with intermediate and 73% (11 of 15) of those with high TS activity responded (P = 0.001). In Cox multivariate regression analysis in which TS was the only significant variable, intermediate and high TS activities were associated with a slow disease progression (P = 0.005) and prolonged survival (P = 0.016) on chemotherapy. In conclusion, for patients with recurrent breast cancer, high tumor TK activity is a significant marker of poor clinical outcome on tamoxifen therapy. Elevated tumor TS activity predicts a favorable outcome for 5-FU-containing polychemotherapy when applied after tumor progression on endocrine therapy.

Plasminogen activator inhibitor-2: prognostic relevance in 1012 patients with primary breast cancer.

The antigen levels of urokinase-type plasminogen activator (uPA) and its inhibitor plasminogen activator inhibitor (PAI) 1, as detected in tumor extracts by ELISA, have been reported to be correlated with a poor prognosis in primary breast cancer. In the present study we have characterized a novel PAI-2-specific ELISA, designed to measure PAI-2 antigen levels in tumor cytosols. We determined PAI-2 antigen levels along with those of uPA and PAI-1 in 1012 routinely prepared tumor cytosols of patients with primary breast cancer (median follow-up, 71 months). In the overall population there was no significant association between the level of PAI-2 and prognosis, while in tumors with high uPA values, PAI-2 (test for trend) was associated with a prolonged relapse-free survival, metastasis-free survival, and overall survival (for all analyses, P < 0.02). In Cox's multivariate analysis for relapse-free survival, metastasis-free survival, and overall survival in tumors with high uPA values (including patient's age, menopausal status, lymph node status, tumor size, estrogen and progesterone receptor status, uPA, and PAI-1), PAI-2 either dichotomized or, as a continuous variable, was independently associated with a favorable relapse-free survival, metastasis-free survival, and overall survival. We conclude that the PAI-2-specific ELISA described herein is well suited for the measurement of PAI-2 levels in cytosols routinely prepared for analysis of steroid hormone receptors. We speculate that PAI-2 may serve as an inhibitor for uPA in human primary breast cancers.

High tumor levels of vascular endothelial growth factor predict poor response to systemic therapy in advanced breast cancer.

Vascular endothelial growth factor (VEGF), a potent angiogenic factor, has been reported to be associated with a poor prognosis in primary breast cancer and in several other cancer types. In the present study, we have measured with ELISA the levels of VEGF in cytosolic extracts of 845 primary breast tumors of patients who developed a recurrence during follow-up. All of the patients received tamoxifen (n = 618) or cyclophosphamide, methotrexate, 5-fluorouracil (CMF) or 5-fluorouracil, Adriamycin, cyclophosphamide (FAC) chemotherapy (n = 227) as first-line systemic therapy after diagnosis of advanced disease. VEGF levels were not related to age or menopausal status but were negatively related to the cytosolic levels of estrogen receptor and progesterone receptor (P < 0.0001). In patients who relapsed within 1 year after primary surgery, tumor VEGF levels were higher than in patients who showed a longer disease-free interval (P = 0.0005). In patients with a first relapse in the viscera, VEGF levels were higher compared with those that relapsed to the bone or soft tissue (P = 0.0004). In univariate analysis for response to first-line tamoxifen therapy, patients with high or intermediate levels showed a poor rate of response, compared with patients with low tumor-VEGF levels (P = 0.0001). Similarly, in multivariate analysis for response to tamoxifen treatment, corrected for age, site of relapse, disease-free interval, and estrogen receptor and progesterone receptor status, VEGF status was an independent predictive factor (P = 0.009). In concordance, higher levels of VEGF were associated with a short progression-free survival and postrelapse overall survival (both, P < 0.0001). On first-line chemotherapy, the rate of response decreased with higher tumor levels of VEGF, both in univariate (P = 0.003) and in multivariate analysis (P = 0.004). Furthermore, higher VEGF levels were associated with a short progression-free survival (P = 0.003) and postrelapse overall survival (P = 0.001). In conclusion, the tumor VEGF level is an important independent marker that predicts a poor efficacy of both tamoxifen and chemotherapy in advanced breast cancer. Knowledge of the tumor level of VEGF might be helpful in selecting individual patients who may benefit from treatments with antiangiogenic agents combined with conventionally used drugs.

The urokinase system of plasminogen activation and prognosis in 2780 breast cancer patients.

The antigen levels of components of the urokinase-type plasminogen activator (uPA) system of plasminogen activation are correlated with prognosis in several types of cancers, including breast cancer. In the present study involving 2780 patients with primary invasive breast cancer, we have evaluated the prognostic importance of the four major components of the uPA system [uPA, the receptor uPAR (CD87), and the inhibitors PAI-1 and PAI-2]. The antigen levels were determined by ELISA in cytosols prepared from primary breast tumors. The levels of the four factors significantly correlated with each other; the Spearman rank correlation coefficients (r(s)) ranged from 0.32 (between PAI-2 and PAI-1 or uPAR) to 0.59 (between uPA and PAI-1). The median duration of follow-up of patients still alive was 88 months. In the multivariate analyses for relapse-free survival (RFS) and overall survival (OS), we defined a basic model including age, menopausal status, tumor size and grade, lymph node status, adjuvant therapy, and steroid hormone receptor status. uPA, uPAR, PAI-1, and PAI-2 were considered as categorical variables, each with two cut points that were established by isotonic regression analysis. Compared with tumors with low levels, those with intermediate and high levels showed a relative hazard rate (RHR) and 95% confidence interval (95% CI) of 1.22 (1.02-1.45) and 1.69 (1.39-2.05) for uPA, and 1.32 (1.14-1.54) and 2.17 (1.74-2.70) for PAI-1, respectively, in multivariate analysis for RFS in all patients. Compared with tumors with high PAI-2 levels, those with intermediate and low levels showed a poor RFS with a RHR (95% CI) of 1.30 (1.14-1.48) and 1.76 (1.38-2.24), respectively. Similar results were obtained in the multivariate analysis for OS in all patients. Furthermore, uPA and PAI-1 were independent predictive factors of a poor RFS and OS in node-negative and node-positive patients. PAI-2 also added to the multivariate models for RFS in node-negative and node-positive patients, and in the analysis for OS in node-negative patients. uPAR did not further contribute to any of the multivariate models. A prognostic score was calculated based on the estimates from the final multivariate model for RFS. Using this score, the difference between the highest and lowest 10% risk groups was 66% in the analysis for RFS at 10 years and 61% in the analysis for OS. Moreover, separate prognostic scores were calculated for node-negative and node-positive patients. In the 10% highest risk groups, the proportion of disease-free patients was only 27 +/- 6% and 9 +/- 3% at 10 years for node-negative and node-positive patients, respectively. These proportions were 86 +/- 4% and 61 +/- 6% for the corresponding 10% lowest risk groups of relapse. We conclude that several components of the uPA system are potential predictors of RFS and OS in patients with primary invasive breast cancer. Knowledge of these factors could be helpful to assess the individual risk of patients, to select various types of adjuvant treatment and to identify patients who may benefit from targeted therapies that are currently being developed.

Complete sequencing of TP53 predicts poor response to systemic therapy of advanced breast cancer.

TP53 has been implicated in regulation of the cell cycle, DNA repair, and apoptosis. We studied, in primary breast tumors through direct cDNA sequencing of exons 2-11, whether TP53 gene mutations can predict response in patients with advanced disease to either first-line tamoxifen therapy (202 patients, of whom 55% responded) or up-front (poly)chemotherapy (41 patients, of whom 46% responded). TP53 mutations were detected in 90 of 243 (37%) tumors, and one-fourth of these mutations resulted in a premature termination of the protein. The mutations were observed in 32% (65 of 202) of the primary tumors of tamoxifen-treated patients and in 61% (25 of 41) of the primary tumors of the chemotherapy patients. TP53 mutation was significantly associated with a poor response to tamoxifen [31% versus 66%; odds ratio (OR), 0.22; 95% confidence interval (CI), 0.12-0.42; P < 0.0001]. Patients with TP53 gene mutations in codons that directly contact DNA or with mutations in the zinc-binding domain loop L3 showed the lowest response to tamoxifen (18% and 15% response rates, respectively). TP53 mutations were related, although not significantly, to a poor response to up-front chemotherapy (36% versus 63%; OR, 0.34; 95% CI, 0.09-1.24). In multivariate analysis for response including the classical parameters age and menopausal status, disease-free interval, dominant site of relapse, and levels of estrogen receptor and progesterone receptor, TP53 mutation was a significant predictor of poor response in the tamoxifen-treated group (OR, 0.29; 95% CI, 0.13-0.63; P = 0.0014). TP53-mutated and estrogen receptor-negative (<10 fmol/mg protein) tumors appeared to be the most resistant phenotype. Interestingly, the response of patients with TP53 mutations to chemotherapy after tamoxifen was not worse than that of patients without these mutations (50% versus 42%; OR, 1.35, nonsignificant). The median progression-free survival after systemic treatment was shorter for patients with a TP53 mutation than for patients with wild-type TP53 (6.6 and 0.6 months less for tamoxifen and up-front chemotherapy, respectively). In conclusion, TP53 gene mutation of the primary tumor is helpful in predicting the response of patients with metastatic breast disease to tamoxifen therapy. The type of mutation and its biological function should be considered in the analyses of the predictive value of TP53.